MARDy : Mycology Antifungal Resistance Database

J.R. was supported by an Antimicrobial Research Collaborative (ARC) early career research fellowship, Imperial College London (RSRO_54990). T.S. and J.M.G.S. were supported by a Natural Environment Research Council grant awarded to MCF (NE/P001165/1).Peer reviewedPublisher PD

Rapid detection of mobilized colistin resistance using a nucleic acid based lab-on-a-chip diagnostic system

The increasing prevalence of antimicrobial resistance is a serious threat to global public health. One of the most concerning trends is the rapid spread of Carbapenemase-Producing Organisms (CPO), where colistin has become the last-resort antibiotic treatment. The emergence of colistin resistance, including the spread of mobilized colistin resistance (mcr) genes, raises the possibility of untreatable bacterial infections and motivates the development of improved diagnostics for the detection of colistin-resistant organisms. This work demonstrates a rapid response for detecting the most recently reported mcr gene, mcr−9, using a portable and affordable lab-on-a-chip (LoC) platform, offering a promising alternative to conventional laboratory-based instruments such as real-time PCR (qPCR). The platform combines semiconductor technology, for non-optical real-time DNA sensing, with a smartphone application for data acquisition, visualization and cloud connectivity. This technology is enabled by using loop-mediated isothermal amplification (LAMP) as the chemistry for targeted DNA detection, by virtue of its high sensitivity, specificity, yield, and manageable temperature requirements. Here, we have developed the first LAMP assay for mcr−9 - showing high sensitivity (down to 100 genomic copies/reaction) and high specificity (no cross-reactivity with other mcr variants). This assay is demonstrated through supporting a hospital investigation where we analyzed nucleic acids extracted from 128 carbapenemase-producing bacteria isolated from clinical and screening samples and found that 41 carried mcr−9 (validated using whole genome sequencing). Average positive detection times were 6.58 ± 0.42 min when performing the experiments on a conventional qPCR instrument (n = 41). For validating the translation of the LAMP assay onto a LoC platform, a subset of the samples were tested (n = 20), showing average detection times of 6.83 ± 0.92 min for positive isolates (n = 14). All experiments detected mcr−9 in under 10 min, and both platforms showed no statistically significant difference (p-value > 0.05). When sample preparation and throughput capabilities are integrated within this LoC platform, the adoption of this technology for the rapid detection and surveillance of antimicrobial resistance genes will decrease the turnaround time for DNA detection and resistotyping, improving diagnostic capabilities, patient outcomes, and the management of infectious diseases

Integrated analysis of patient networks and plasmid genomes reveals a regional, multi-species outbreak of carbapenemase-producing Enterobacterales carrying both blaIMP and mcr-9 genes

Background Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of IMP-encoding CPE amongst diverse Enterobacterales species between 2016 and 2019 across a London regional network. Methods We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE positive patients. Genomes of IMP-encoding CPE isolates were overlayed with patient contacts to imply potential transmission events. Results Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, E. coli); 86% (72/84) harboured an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68/72). Phylogenetic analysis of IncHI2 plasmids identified three lineages showing significant association with patient contacts and movements between four hospital sites and across medical specialities, which was missed on initial investigations. Conclusions Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multi-modal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks

Fusarium: more than a node or a foot-shaped basal cell

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae. Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae. Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium. Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae. Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae (e.g., Cosmosporella, Macroconia, Microcera). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium. To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa (act1, CaM, his3, rpb1, rpb2, tef1, tub2, ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [F. dimerum species complex (SC)], Cyanonectria (F. buxicola SC), Geejayessia (F. staphyleae SC), Neocosmospora (F. solani SC) or Rectifusarium (F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org)

Molecular epidemiology of azole-resistant Aspergillus fumigatus in respiratory infections

Aspergillus fumigatus is a ubiquitous ascomycete mould and the primary cause of aspergillosis which varies in severity and clinical presentation. Triazoles have been the most widely used antifungal agents in prophylaxis and treatment of Aspergillus-related infections. Since the late 2000s there has been a steady increase in the number of reported resistance to azole antifungals in A. fumigatus, causing a major clinical concern with subsequent treatment failure among some patients. This PhD aimed to clarify the epidemiology of azole-resistant A. fumigatus at a regional level in West London. Firstly, the prevalence of azole-resistance in a large collection of clinical A. fumigatus isolates collected over a 17 year period (1998 - 2017) was determined, which surprisingly found a low level of resistance among mixed patient population. Conversely, a two-year (2015 - 2017) passive surveillance of isolates obtained from a specialist cardio-thoracic centre located in the same region, revealed an alarmingly high prevalence of resistance mostly among patients with cystic fibrosis. Secondly, to evaluate the application of whole-genome sequencing (WGS) in interrogation of azole-resistant A. fumigatus, a global panel of 24 isolates was analysed. WGS demonstrated that SNP analysis was able to accurately identify polymorphisms in the cyp51A gene conferring resistance to triazole antifungals. The WGS further associated to a globally panmictic population in A. fumigatus. In London, WGS revealed that environmentally-driven TR34/L98H, G54W and P216L were common alterations in cyp51A. A novel TR34/L98H/T289A/G448S polymorphism was discovered here causing a pan-azole-resistant phenotype. Phylogenomic analysis of sequenced genomes including London isolates, showed two novel major clades; one clade contains azole-resistant A. fumigatus with TR34/L98H allele, while the second clade consists of most azole-susceptible isolates. Finally, this PhD characterises the phenotypic heterogeneity of A. fumigatus and showed that acidic pH tolerance exists among some clinical A. fumigatus isolates. Furthermore, phenotypic switching accompanied with enhanced growth rate found in one azole-resistant isolate indicates that azole-resistant A. fumigatus has high fitness in azole-free environments and confirms that sexual reproduction leads to transmission of TR34/L98H allele in the absence of exposure to medical or fungicidal azole agents. Overall these findings indicate that prevalence of azole-resistance is linked to specific patient populations in regional level. Presence of TR34/L98H among azole-resistant isolates suggests a spillover of environmentally acquired antifungal resistance into susceptible patient cohorts.Open Acces

Sexual transmission of intestinal parasites in men who have sex with men.

Direct oral-anal sexual contact is a common practice among men who have sex with men (MSM) and is implicated in the transmission of various enteric pathogens including intestinal parasites. The present study reviewed data on the sexual transmission of intestinal parasites among MSM, and highlighted advances in the diagnosis of such infections. The emergence and spread of intestinal parasites is of public health concern particularly in the homosexual community. Intestinal parasitic infection should be considered in the differential diagnosis of gastrointestinal disease in this population. Combination of traditional diagnostic procedures with implementation of testing based on novel molecular methods in the accurate identification of intestinal parasites is important so that early intervention and control of infection is facilitated

Quinolone Resistance in Oligella Urethralis-Associated Urinary Tract Infection

The importance of oxidase-positive, nonfermentative, Gram-negative bacilli in a variety of pathological processes is becoming increasingly important. Oligella urethralis is an organism which is normally isolated as a commensal from the genitourinary tract. We have described one case of urinary tract infection caused by this organism. This isolated organism is found to be resistant to quinolones, while relatively sensitive to a wide variety of antibiotics. These findings clearly indicate that this organism may be an opportunistic pathogen and that extensive application of quinolones may provide a selective pressure for the emergence of resistance. We believe that this is the firs report of isolation and quinolone resistance in Oligella in Iran

Fungal Nomenclature: Managing Change is the Name of the Game

Fungal species have undergone and continue to undergo significant nomenclatural change, primarily due to the abandonment of dual species nomenclature in 2013 and the widespread application of molecular technologies in taxonomy allowing correction of past classification errors. These have effected numerous name changes concerning medically important species, but by far the group causing most concern are the Candida yeasts. Among common species, Candida krusei, Candida glabrata, Candida guilliermondii, Candida lusitaniae, and Candida rugosa have been changed to Pichia kudriavzevii, Nakaseomyces glabrata, Meyerozyma guilliermondii, Clavispora lusitaniae, and Diutina rugosa, respectively. There are currently no guidelines for microbiology laboratories on implementing changes, and there is ongoing concern that clinicians will dismiss or misinterpret laboratory reports using unfamiliar species names. Here, we have outlined the rationale for name changes across the major groups of clinically important fungi and have provided practical recommendations for managing change